The plasmid we designed contains an insert of fifteen ACPs respectively, and ligated with the pET plasmid. The pET plasmid contains kanamycin resistance gene, SUMO-tag, 6x His tag and a Multiple Cloning Site. Apart from the ACP gene, a cell-penetrating peptide (CPP) is also included in some inserts.
cell-penetrating peptide (CPP)
A sequence added to the ACPs’ ending point. It functions to prompt a cell to translocate the protein to the cell membrane, increasing the expression of ACPs
Multiple Cloning Site
A region of DNA within a plasmid. It contains multiple unique and contiguous restriction enzyme recognition sites, so it provides flexibility in the choice of restriction enzymes. In our plasmid design, 2 cut sites, (NdeI and SacI) located before and after SUMO are used to remove the SUMO part and insert the ACPs with cell-penetrating peptide (CPP). NheI is retained for further modification at 5’ end if needed. KpnI is located in pET-plasmid after SacI for the 3’ end modification if necessary.
6-His tag
It is one of the simplest and most widely used purification tags and commonly used in the production of recombinant proteins. These residues readily coordinate with transition metal ions immobilized on beads or a resin for purification.
Before digestion and ligation, all plasmids are amplified by DH5-alpha bacterial culture and purified by miniprep. Next, SacI and NdeI were used to cut open the pET plasmid and all ACP inserts and both fragments were ligated to form different pET-ACPs respectively.
The recombinant plasmid first was transformed in DH5-alpha for cloning. And then, the plasmid was transformed in BL21 after the ‘Step 3: Colony PCR’.
To confirm whether the bacteria in colony get the correct plasmid, colony PCR was conducted. We designed some primers for colony PCR. Details show below
A single colony was picked up and incubated in 20ml LB broth containing kanamycin(1X) at 37℃ with 150rpm shaking overnight. A total of 6ml incubated cell culture was then incubated into 240ml LB broth containing kanamycin(1X) at 37℃ with 150rpm shaking until the OD600 reached 0.6. Then, 0.4mM IPTG was used to induce the protein expression for 24hrs at 20℃. This step repeated seven times for seven AMPs expression.
SDS-PAGE would be conducted to test did the expression be induced successfully and estimate the protein concentration.
If target bands have been shown in SDS-PAGE results, the corresponding supernatant samples will undergo His-tag purification. After that, another SDS-PAGE will be conducted to confirm whether the ACPs were purified.
To increase the concentration of the peptide. We used the Amicon Ultra-0.5 mL Centrifugal Filters for Protein Purification and Concentration. 500 µL of sample were added to the Amicon Ultra filter device, which is then placed into the centrifuge rotor. The device was spun at 14,000×g for 20mins. After that, the assembled device was removed from the centrifuge and the Amicon Ultra filter device was separated from the microcentrifuge tube. To recover the concentrated solute, the Amicon Ultra filter device was placed upside down in a clean microcentrifuge tube and placed in centrifuge. To transfer the concentrated sample from the device to the tube, it is spun for 2 minutes at 1,000×g.
The human epithelial lung cancer cell line A549 was purchased from the American Type Culture Collection (ATCC, USA). A549 were cultured in Dulbecco’s Modified Eagle Medium F12/DMEM (Thermo Fisher Scientific, USA). A549 cells were cultured and seeded on a T75 flask at a density of 2.1x10^6 cells. The previous culture medium was removed. TrypLE Express Enzyme (ThermoFisher Scientific, USA) was added and cells were incubated in a CO2 incubator until all adherent cells were removed from the lower surface of the plate. Transfer the content to a falcon tube and centrifuge it at 1000 rpm for 5 minutes. The supernatant was removed, while the cell pellet was then resuspended by fresh culture medium.
For cell counting, 10μl cell suspension and 10μl Trypan blue solution was mixed well. The mixture is loaded onto a hemocytometer and observed with EVOS XL Core Imaging System (Thermo Fisher Scientific, USA) to count the number of cells in four grids. The desired cell seeding density is 5000 cells per well. The required amount of cell suspension was mixed with the corresponding volume of culture medium in a falcon tube. The mixture was added into a reagent reservoir and transferred to the 96-well plate to perform cell seeding at a density of 5000 cells per well. The outer wells were filled with 200μl sterilized water to avoid disturbance of cells by excessive evaporation [1].
After two days of growth, previous growth medium was removed and cells were treated with new medium with different concentrations or combinations of drugs to investigate their effect on cancer cell viability. Below table shows the detail.
As a preliminary cytotoxicity test of AC-P19, we only observe the morphology of A549 cells by EVOS XL Core Imaging System (Thermo Fisher Scientific, USA) to ensure it AC-P19 is work in our culture system. For later cytotoxicity test, Cell Counting Kit-8 (CCK-8) would be used to measure cell viability.
We repeat step 4 to express four Cordyceps militaris peptides (C-ori, C-rds, CTP-ori and CTP-rds).
We repeat step to purify and concentrate four Cordyceps militaris peptides (C-ori, C-rds, CTP-ori and CTP-rds).
We repeated step 6(i) to culture A549 cell.
After two days of growth, previous growth medium was removed and cells were treated with new medium with different concentrations or combinations of drugs to investigate their effect on cancer cell viability. Below tables show the detail.
Cell Counting Kit-8 (CCK-8) measures cell viability by correlating the production of colored formazan dye to the number of living cells in culture. Dehydrogenases from viable cells convert WST-8 into a formazan dye, which produces an easily measurable color change that can be quantified in a microplate reader at 450 nm.
After drug incubation, 10μl of CCK-8 reagent was added directly to the wells. The plates were then incubated for a specified period, within 30 minutes to one and a half hours, to allow sufficient time for the color to develop.
After incubation, the absorbance of each well was recorded using a microplate reader with a wavelength of 450 nm. Since the intensity of the color is proportional to the number of viable cells, the cell viability of cancer cells treated with different concentrations of drugs can be compared.
In the measurement that involved 3D spheroid, 100μl of medium was removed and 10μl of CCK-8 reagent was added directly to the wells. The plate was shaken to ensure even distribution of newly formed formazan dye [2].
We repeated step 1-5 to get purified peptides from GM E. coli (BL21).
We repeated step 9 to test the effect of those peptides to A549 cell line. Below table shows the detail of drug treatment.
We repeated step 9 to test the effect of the most efficiency ACP (KAPI) to A549 cell line and BEAS-2B cell line. Below tables show the detail of drug treatment.
We repeated step 9 to test the combination effect of the most efficient ACP (KAPI) and cisplatin to A549 cell line
We followed below procedure for growing Multi-Cellular Tumor Spheroid (MCTS). 1. 100μl of cell suspension was transferred to each well of a U-bottom 96-well Nunclon Sphera microplates (Thermo Fisher Scientific) [3] 2. The plate is centrifuged at 2500 rpm for 5 minutes [4] to help the cells clump at the bottom of the well, facilitating uniform spheroid formation (Figure 3).
To maintain consistency in the spheroids selected for the analysis of cancer growth, tier 1 and tier 2 screenings are conducted. In tier 1 screening, the morphology and sphericity of the spheroid are assessed via live imaging using the inverted microscope. The sphericity of the spheroids is assessed and those with low sphericity (Figure 4) were excluded from the downstream processing. After tier 1 screening, the spheroid with a similar morphology will proceed to tier 2 screening (Figure 5).
In the tier 2 screening, the software tools ImageJ and ReVISP are employed to assess their diameter and volume, respectively. providing data for tier 2 screening.
To begin with, the diameter of the spheroid is assessed by Image J. It is a public domain software used to process and analyze scientific images.[5] Related procedures are as follows:
Image:
Next, the volume of the spheroid is estimated by ReVISP. ReVISP is a 3D volume rendering MATLAB tool for multicellular spheroid to reconstruct the 3D shape of multicellular spheroids and for the estimation of volume by counting the voxels (3D pixels) fully included in the 3D surface. [6][7]. The related procedure is as follows:
ReVISP:
1. Draw the spheroid circumference and create mask of spheroid through ImageJ. Save the mask as a tiff. Image.
2. Input the tiff. image of the spheroid mask into ReVISP to reconstruct the 3D shape of the spheroid. (Figure 8)
3. The 3D volume of the spheroid is calculated
Data of diameter and volume are obtained and their CV is calculated. (Figure 9)
In order to assess the reliability of the data obtained, Coefficient of variation (CV) was calculated. CV value is a measure of precision and is equal to the standard deviation quantity divided by the mean quantity of a group of replicates. CV is commonly used to assess the reproducibility of in vitro models, and CV scores of < 20% variation are considered acceptable. The calculated CV value for spheroid diameter is 15.3% while that of volume is 6.14%. Each value is acceptable and hence the spheroids are used for downstream processing. Also, spheroids’ data beyond 2 SDs from the mean are considered outliers. Those will be excluded from downstream processing. Through tier 1 screening and the application of ImageJ and REVISP, consistent spheroid size and uniformity can be maintained for more precise measurement in cytotoxicity test.
In our hypothesis, KAPI binds to PDEδ and stops activating KRAS and its subsequent signaling pathway:[8][9]
KRAS normally binds with GDP in an inactivated state. When the receptor EGFR receive extracellular growth factors such as EGF, it transmits signals to receptors, the SOS, and interacts with the KRAS-GDP complex leading to the replacement of GTP. This molecular switch controls multiple signaling pathways, including the MARK/ERK pathway, which controls cell proliferation; PI3K-AKT-mTOR pathway, which promotes cell survival and growth.
To verify the molecular mechanism of KAPI, reverse transcription‑quantitative (RT‑q) PCR is carried out. Cells are seeded at a 6-well plate with a density of 0.3 x 106 cell per well. After two days of cell growth, the old medium was removed and the new medium with 0μM, 25μM and 50μM ACP5 (KAPI), were added into the well respectively. After treatment for four hours, TRIzol (Biosharp Life Sciences) was used to extract total RNA of different treated group. The absorbance values at wavelengths of 260 and 280 nm of the total RNA were measured by Nano‑Drop ND‑1000 spectrophotometry, and the OD260/OD280 ratio was calculated to make sure the RNA sample is of good quality (ratio >1.80) (Figure 11).
A reverse transcription reaction (Biosharp Life Sciences) was carried out on a PCR amplification apparatus to synthesize the cDNA template.
Real-time qPCR was then performed with LightCycler® 480 System (Roche Diagnostics) using SYBR Green qPCR Mix (Biosharp Life Sciences). (Figure 12 and 13)
The gene expression levels of EGFR1, BRAF, Pl3K is checked, with GAPDH2 as reference. The primer sequences (synthesized by BGI Tech Solutions) are as follows:
The target mRNA abundance in each sample was normalized to its reference level as ΔCt= Ct (signalling pathway gene)-Ct (house-keeping gene). The Ct value is the quantification cycle number and ΔΔCt is the difference of ΔCt(samples) with the ΔCt (control, KAPI=0μM). 3 and 5 replicas were performed respectively. The reduction of mRNA level is expressed as a percentage and calculated with the formula (2^-ΔΔCt).
1. Thermo Fisher Scientific. Preventing edge effect during extended culture in microplates with a unique perimeter moat design. https://assets.thermofisher.com/TFS-Assets/LCD/Application-Notes/COL31874-EDGE-App-Note.pdf
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3. Thermo Fisher Scientific. Disease model generation: 5 steps to a 3D cancer spheroid model. https://assets.thermofisher.cn/TFS-Assets/BID/brochures/5-steps-3d-cancer-spheroid-brochure.pdf
4. Oner E, Gray SG, Finn SP. Cell Viability Assay with 3D Prostate Tumor Spheroids. Methods Mol Biol. 2023;2645:263-275. doi: 10.1007/978-1-0716-3056-3_17. PMID: 37202626.
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7. F. Piccinini, et al., Single-image based methods used for non-invasive volume estimation of cancer spheroids: a practical assessing approach based on entry-level equipment. Computer Methods and Programs in Biomedicine, 135:51-60, 2016.
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